Method for treating exposed dental pulp

ABSTRACT

The invention relates to the application of a composition containing recombinant amelogenin and propylene glycol alginate (“PGA”) on to cut dentin tubules followed by installation of a definitive restorative in a single visit.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of dentistry and dentalreconstruction. More specifically, to preserving the natural, livingteeth of a subject and avoiding procedures producing dead teeth or theneed for prosthetic implants.

Description of the Related Art

The exposure or near exposure of living or vital pulp inside of a toothto caries and other forms of decay, from trauma, such as from a brokenor cracked tooth, or to a dental procedure that erodes the enamel anddentin can cause severe pain and the risk of inflammation and infectionwhich can destroy the living or vital pulp. Unlike mechanical damage toa tooth in which pre-existing inflammation is not present, penetrationof the pulp by caries results in microbial invasion of the pulp,resulting in pulpal inflammation and leaving the pulp less able torespond to the insult and heal; Murray P E, Windsor L J, Smyth T W,Hafez A A, Cox C F. Analysis of pulpal reactions to restorativeprocedures, materials, pulp capping, and future therapies. CriticalReviews in Oral Biology & Medicine. 2002; 13(6):509-520; NorthwestPrecedent. A survey of practitioner preference in direct pulp cappingmaterials, 2007.

The morbidity associated with treating pulp exposures is consequentialand often requires expensive and painful procedures such as root canalor endodontic therapy, tooth extraction, or prosthetic replacement of atooth. However, the loss of vital pulp in a tooth results in the loss oftooth sensation, in the inability of a tooth to repair itself, and in adecreased lifespan for the tooth.

As an alternative to endodontic therapy or extraction or replacement ofa damaged tooth, the tooth may be capped. Pulp capping involves placinga medicament on top of the exposed pulp tissue in an attempt to promotepulp healing and dentin repair; see tooth anatomy in FIG. 11 (priorart). A number of materials have been suggested for use in direct pulpcapping, however, no one material seems to enjoy a significantpreference among practitioners; Paine M L, White S N, Luo W, Fong H,Sarikaya M and Snead M L. Regulated gene expression dictates enamelstructure and tooth function. Matrix Biol 2001; 20:273; Zeichner-DavidM. Is there more to enamel matrix proteins than biomineralization?Matrix Biol. 2001; 20:307). An ideal capping procedure or cappingmaterial would not only form a dentin bridge over the breach caused bycaries or injury, but preserve the vitality and integrity of the livingpulp inside of the tooth. Ideally, a capping procedure would onlyrequire a single visit to a dentist to repair and cap the tooth.

There are two types of capping: indirect capping and direct capping.When caries are in close proximity to the pulp, an indirect pulp cap canbe performed. In this procedure, much of the infected and affecteddentin is removed without exposing the pulp in a vital, healthy tooth.This is followed by the placement of a medicament (such as calciumhydroxide or a bioactive material) in order to promote the formation oftertiary dentin. Stepwise indirect capping involves cavity preparationto remove all carious enamel, but leaves carious dentin in placeadjacent to the pulp chamber. A medicament is placed over the remainingcaries, a provisional restoration is placed and the patient is scheduledfor a second appointment. At the second visit, the provisionalrestoration and remaining caries are removed, and a definitiverestoration is placed. The goal of a stepwise approach is to bothpromote the formation of tertiary dentin and change the environmentwithin the tooth to prevent further progression of the carious lesion.

With a single-visit indirect pulp cap, as much of the carious lesion aspossible is removed without inducing a pulpal exposure. A medicament isplaced in the deepest aspect of the preparation, and a definitiverestoration is placed. Unlike the stepwise approach, a single-visitexcavation does not require a second visit to restore the tooth, andthere is no risk of an inadvertent pulpal exposure when re-entry of thetooth occurs.

As long as the pulp is vital and asymptomatic, conventional indirectcapping procedures can leave some decay near the pulp but cover the areawith a bio-compatible material, such as a dentin bonding agents, resinglass ionomers, or calcium hydroxide, can stimulate the repair of thedentin and inhibit further decay. This incomplete removal of decay isnot considered detrimental as long as the filling material completelyseals the dentin from the bacterial environment of the oral cavity.However, this residual material can negatively impact the ability of thetooth to heal itself or regenerate.

A direct pulp cap is done on permanent teeth when the removal of deepdecay results in exposing the pulp. If the pulp appears infected orsymptomatic, a dentist may decide that removal of the living pulp is thebest treatment option, for example as part of a root canal procedure. Insituations where the pulp is vital and healthy, capping exposed pulptissue with a material, such as calcium hydroxide, and providing a goodseal with the filling material may solve the problem and prevent theneed for further endodontic treatment. However, placement ofconventional materials such as calcium hydroxide or mineral trioxideaggregate (“MTA”) in indirect or direct capping procedures can alsonegatively impact the ability of a tooth to heal or regenerate as shownherein by the inventors.

Materials used for direct and indirect capping procedures includecalcium hydroxide and mineral trioxide aggregate (“MTA”) which isprimarily calcium oxide in the form of tricalcium silicate, dicalciumsilicate and tricalcium aluminate. These materials serve as a reservoirfor calcium hydroxide and can provide a type of seal at the site of pulpexposure. However, MTA is highly soluble and can darken a treated toothdue to presence of iron in a gray MTA formulation. Moreover, it requiresa second dental appointment for placement of a definitive restoration;Camilleri J, Montesin F E, Di Silvio L, Pitt Ford T R. The chemicalconstitution and biocompatibility of accelerated Portland cement forendodontic use. International Endodontics Journal. 2005; 38:834-842;Aeinehchi M, Eslami B, Ghanbariha M, Saffar A. Mineral trioxideaggregate (MTA) and calcium hydroxide as pulp-capping agents in humanteeth: A preliminary report. International Endodontics Journal. 2002;36:225-231; Tomson P, Grover L, Lumley P, Sloan A, Smith A, Cooper P.Dissolution of bio-active dentine matrix components by mineral trioxideaggregate. Journal of Dentistry. 2007; 35:636-642; de Souza Costa C,Duarte P, de Souza P, Giro E, Hebling J. Cytotoxic effects and pulpalresponse caused by a mineral trioxide aggregate formulation and calciumhydroxide. American Journal of Dentistry. 2008; 21:255-261; T J Hilton.Keys to Clinical Success with Pulp Capping: A Review of the Literature.Oper Dent.2009 September-October; 34(5): 615-62.

Mineral trioxide aggregate (MTA) is made of hydrophilic fine particlesthat harden in the presence of moisture or blood. It requires a workingtime of about 5 min and a hardening time that, varies from 2 h and 45min to 4 h according to the density of the air entrapped during mixingand the dampness of the receiving site. The long, hardening time reducesinternal tensions and the incidence of marginal infiltration, but itforces to definitively fill the tooth in a subseqeuent dental visit orsitting.

Many conventional dental materials including M′I′A′ have distinctirritability and it has been observed that they exert adverse effects ondifferent body organs. MTA contains heavy metals and releases arsenicand it has been found that MTA contained levels of arsenic that exceededthe levels (2 mg kg_1 of cement) recommended by InternationalStandardization Organization (2007). When arsenic came in contact withblood during surgery, it was absorbed and carried by red blood cells,later leaving the blood stream and deposited in the liver and kidney. Ifthe liver is not able to metabolize the arsenic, it becomes toxic andcauses damage even in sublethal concentrations.

In contrast, as shown by the inventors r-amelogenin protein isbiocompatible, it is composed of the same protein secreted in the oralcavity and it does not release toxic materials. Even if some particlesbecome trapped in the pulp tissue, it will not cause adverse reactions.The pulp tissue capped with amelogenin proteins always restored andpreserved the authentic pulp architecture in addition to forming adentin bridge. No other capping material showed similar results

Amelogenins are a family of proteins rich in proline, histidine,glutamine and leucine. Amelogenins are involved in forming a scaffoldfor mineral deposition and for production of cementum, dentin, andenamel; participate in hydroxyapatite crystallite formation, and form amatrix that regulates the subsequent crystallite growth. Moradian-OldakJ. Assembly, processing and control of crystal morphology Matrix Biology2001; 20: 293); Fincham A G, Moradian-Oldak J, Simmer J P, Sart P, Lau EC, Diekwisch T and Slavkin H C. Self assembly of a recombinantamelogenin protein generates supramolecular structures. J Struct Biol1994; 112:103.

Prior attempts have been made to use enamel matrix proteins Theseinclude the use recombinant amelogenin protein to induce apical (at baseof a root of a tooth) closure and pulp regeneration in non-vitalimmature teeth in canines (Mounir, et al., J. Endod. 2016,42(3):402-412); for apexification and root canal treatment of thepremolar teeth of young dogs with amelogenin and PGA (Mounir, et al.,US20140023979A1), and for regeneration of dentin (Lyngstadaas, et al.,U.S. Pat. No. 7,304,030). Prior methods, such as those described byMounir only cut the enamel, for example, Mounir only cuts the enamel incervical region 7 and does not touch or cut dentin. Such procedures donot make deep cuts in the dentin which expose the inside of the dentinaltubules to the matrix material which penetrate the cytoplasm of thetubule to reach the odontoblast layer and the pulp tissue.

However, mixtures of amelogenin and PGA have not been previously usedfor dental capping or evaluated against conventional materials such asMTA. Surprisingly, the inventors found that a mixture of amelogenin andPGA placed on open healthy dentinal tubules accelerated dentin bridgeformation and replenishment of dental pulp compared to the conventionalmaterial MTA. This procedure can form a dentin bridge, preserveauthentic pulp and induce regenerative of pulp tissue in previouslyempty canals. Moreover, the inventors have found that a mixture ofamelogenin and PGA can be effectively used for capping proceduresrequiring only a single visit.

BRIEF SUMMARY OF THE INVENTION

The invention relates to applying a composition containing recombinantamelogenin and propylene which is preferably performed in a singlevisit. This simplified procedure regenerates dentin and forms a dentinbridge over a breached pulp chamber and overtime provides anincreasingly solid dentin foundation for the restorative. This methodregenerates dentin faster, and replenishes vital pulp without leavingempty spaces or voids in pulp tissue better, than conventionalprocedures using capping materials such as MTA. Nonlimiting embodimentsof the invention include the following.

A method for indirect or direct capping of a tooth in a subject having adamaged tooth that comprises, consists essentially of, or consists ofapplying a composition comprising amelogenin and propylene glycolalginate (“PGA”) to a surface of a pulp-containing tooth comprising opendentinal tubules or exposed pulp. In many embodiments of the invention,deep cuts are made to dentin which expose the inside of the dentinaltubules to the matrix material which penetrate the cytoplasm of thetubule to reach the odontoblast layer and the pulp tissue. In oneembodiment this method indirectly caps a damaged tooth by applying thecomposition to open dentinal tubules. In another embodiment, this methoddirectly caps a damaged tooth by applying the composition to open dentintubules and/or to exposed pulp. In preferred embodiments a finalrestorative is applied over the surfaces of the teeth treated with theamelogenin and PGA and can be applied during a single visit or on thesame day or within a span not exceeding 24 hours to the dentist. It isnot necessary to perform a stepwise cap which requires a second dentalvisit. Moreover, unlike conventional direct capping procedures usingcalcium hydroxide or MTA, the method as disclosed herein usingamelogenin and PGA provides for rapid formation of a dentin bridge overa breach or near breach of the pulp chamber of a tooth and providessuperior regeneration of vital pulp tissue.

In some embodiments of this method a subject's tooth has been exposed tomechanical trauma not associated with a dental procedure; or has beenexposed to mechanical trauma associated with a dental procedure. It maybe used to treat or repair various forms of dental trauma includingenamel infarction, enamel fracture, enamel-dentine fracture,enamel-dentine fracture involving pulp exposure, or injuries includingroot fracture.

The composition used in the method disclosed above can containamelogenin which comprises an amino acid sequence that is at least 75,80, 85, 90, 95, 96, 97, 98, 99 or 100% identical to the amino acidsequence of SEQ ID NO: 2, 4 or 6 or a sequence that has at least 75, 80,85, 90, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NOS: 2, 4or 6 or a fragment thereof having at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180or >180 contiguous amino acid residues of the sequence. Preferably, thesubject is a human and can be treated with a composition containinghuman amelogenin or a fragment thereof having at least 30 contiguousamino acid residues of the human amelogenin. However, in view of theconservation of amelogenin sequences, treatment with murine or othertypes of amelogenins may also be performed.

The composition used in the methods disclosed herein may contain 10, 20,30, 40, 50, 60, or >60 mg of amelogenin per 1 cc of propylene glycolalginate vehicle, preferably from about 40 mg per 1 cc of PGA vehicle,or more preferably about 30 mg of amelogenin per 1 cc of PGA vehicle. Insome embodiments, the composition will comprise, consist essentially of,or consist of amelogenin and PGA. The use of recombinant amelogeninpermits one to eliminate the presence of other biological componentsthat co-purify when amelogenin is isolated from a natural source thusproviding an easily standardizable composition for use in dental ormedical procedures.

Amelogenin may be present in a composition of the invention at aconcentration ranging from 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10,20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/cc in combination with avehicle containing PGA. This range includes all intermediate values andsubranges.

A PGA vehicle may contain a concentration of PGA ranging from about0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5 or >5 wt % PGA in wateror other aqueous solution, preferably from about 2 to about 3 wt % basedon the total weight of an aqueous solution containing the PGA in orderto provide sufficient viscosity and fluidity to be easily combined withamelogenin and applied to dental surfaces. This range includes allintermediate values and subranges. In one embodiment, the PGA vehiclecontains 20-40 mg of PGA in 1 cm³ saline, more preferably about 30 mg ofPGA in 1 cm³ saline.

In other embodiments, the composition used in the methods disclosedherein will further comprise a remineralization agent selected from thegroup consisting of a material providing calcium ions, a materialproviding phosphorous ions, or a material providing fluoride ions. Thesematerials include, but are not limited to calcium phosphates such ashydroxyapatite, tricalcium phosphate, or calcium monohydrogen phosphate,calcium hydroxide, calcium carbonate, arginine phosphate, sodiummonofluorophosphate, or other salts containing calcium, phosphorous orfluoride ions. It may also include at least one non-amelogenin enamelmatrix protein which may be purified from a natural source, such as fromporcine teeth or recombinantly expressed.

In other embodiments the composition further comprises at least onebacteriostatic or bactericidal agent such as amoxicillin, metronidazole,penicillin, clarithromycin, or clindamycin.

The methods disclosed herein are preferably used on subjects is in needof regeneration of a dentin bridge and/or in need of preservation orregeneration of vital pulp without voids within the pulpal chamber ofthe tooth, such as subjects wishing to retain their living teeth and whoopt to not undergo destructive dental procedures such as root canalsurgery which removes and destroys living pulp.

In one embodiment, the subject is suffering from a subject sufferingfrom amelogenesis imperfecta and said composition comprises a wild-typeamelogenin to compensate for problems caused by the expression orpresence of a mutant form of amelogenin.

Another embodiment of the invention is directed to a compositioncomprising human amelogenin and polyglycol alginate in a ratio of about10 to 60 mg, preferably from about 20 mg to 40 mg amelogenin per ml ofpolyglycol alginate vehicle, in combination with (i) MMP-9, MMP-20,kallikrein or at least one other protease that cleaves or digestsamelogenin; and/or in combination with (ii) amelogenin that has been atleast partially digested by MMP-9, MMP-20, kallikrein 4, or at least oneother protease that cleaves or digests amelogenin. A protease may be atany concentration sufficient to cleave amelogenin or to gradually cleavean applied dose of amelogenin over a period of 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more weeks. Representative concentrations include 0.001, 0.002,0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 or 2 wt % based on the totalweight of the amelogenin and PGA composition. In some embodiments,proteases may be incorporated into a timed-release or modified-releasedosage form to release the protease over a period of time such as over4, 5, 6, 7, 8, 9, or 10 weeks.

An amelogenin and PGA composition as disclosed herein may include leastone non-amelogenin enamel matrix protein, or may include aremineralization agent selected from the group consisting of a materialproviding calcium ions, a material providing phosphorous ions, or amaterial providing fluoride ions.

Another embodiment of the invention is directed to a kit for applying acomposition comprising amelogenin and PGA comprising amelogenin, PGA, adevice for mixing and/or applying the composition to open dentin tubulesor to a surface having exposed pulp, and directions for use.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1. Appearance of teeth treated with amelogenin-PGA after 10 days;

FIG. 2. Appearance of teeth treated with amelogenin-PGA after 10 days;

FIG. 3. Appearance of teeth treated with amelogenin-PGA after 2 months;

FIG. 4. Appearance of teeth treated with amelogenin-PGA after 2 months;

FIG. 5. Appearance of teeth treated with mineral trioxide aggregate(“MTA”) after 10 days;

FIG. 6. Appearance of teeth treated with mineral trioxide aggregate(“MTA”) after 10 days;

FIG. 7. Appearance of teeth treated with MTA at after 2 months;

FIG. 8. Appearance of teeth treated with MTA at after 2 months;

FIG. 9. Appearance of teeth treated with MTA at after 2 months;

FIG. 10. Graph depicting differences at 10 days between teeth cappedwith amelogenin-PGA or by MTA.

FIG. 11. Diagram of a healthy human molar showing the enamel, cementum,pulp, and dentin which make up the structure, as well as the surroundingtissues. Prior art. Author: K. D. Schroeder; Human tooth diagram-en.svgfrom Wikimedia Commons; License: Creative Commons Attribution-ShareAlike4.0.

Abbreviations appearing in the figures: BD: dentin bridge; D: dentin; P:pulp, od: odontoblasts, V: voids; By: blood vessels; PD: predentin; PS:pulp space.

DETAILED DESCRIPTION OF THE INVENTION

A number of materials have been used in direct pulp capping procedures.However, conventional capping materials can negatively impact dentineregeneration and pulp tissue which are exposed during a cappingprocedure. Previously no one pulp material could induce the formation ofdentin bridge and preserve vital pulp tissue exposed during a dentalprocedure.

The present invention provides an amelogenin composition that inducesdentin bridge formation while simultaneously facilitating dentineregeneration and preserving pulp tissue thus avoiding the drawbacksassociated with conventional capping materials such as calcium hydroxideand MTA. The invention also relates to a method for preparing the activeenamel matrix material for the preparation of a pharmaceuticalcomposition capable of regenerating dentin and preserving pulp vitalityfollowing dental procedures involving exposure of vital dental pulptissue.

Advantageously, the amelogenin-PGA composition and its method of use forprotecting and restoring viability to damaged teeth advantageouslyrelate to the characteristic that amelogenin and PGA are easily mixedand applied. The invention provides a natural way to repair and restoreviability in damaged teeth because amelogenin is a protein involved inthe natural growth and enamelization of teeth and thus can interact withother natural components of teeth without the high risk of toxicityassociated with some artificial materials. For example, the inventorshave shown that amelogenin induces minimal inflammatory reactions whenplaced over exposed pulp tissue and preservation rather than damage topulp tissue. The inventors further showed that amelogenin induceshealing of the damaged tooth by inducing formation of tertiary dentinemineralized tissue barrier between the pulp chamber and overlying dentalstructures.

The invention preserves the underlying pulp tissue which becomescontinuous with no internal voids and fills the complete pulp chamberand root canal space in contrast to similar treatments with MTA and thepulp regenerated after treatment with amelogenin-PGA composition appearsto the same or similar to original pulp. The inventors demonstrate thata mixture of amelogenin and PGA can be effectively used as a dental pulpcapping material which quickly regenerates a dentin bridge over a breachor near breach in the pulp chamber of a tooth. They also show thatcapping with a mixture of amelogenin and PGA provides a superior degreeof protection to pulp tissue in a damaged and capped tooth and preventsthe formation of voids seen in teeth capped with conventional agentslike MTA. The method according to the invention preserves pulp tissueand a tooth's vitality as well as its ability to sense heat or cold andother sensations and respond to such stimuli. Preservation of the pulptissue preserves the tooth's ability to repair itself, prevents theweakening of the tooth structure, such as weakening caused by root canalsurgery, that increase the risk of fracture and loss of a repairedtooth. Moreover, the method as disclosed herein prevents pulpinflammation (pulpitis) and subsequent irreversible pulpitis anddegeneration of the dental pulps that would otherwise require additionaldental treatments such as pulpectomy, pulpotomy or root canal treatments(“RCT”).

Subjects of treatment. A subject is typically a person with dentaltrauma, especially trauma which exposes vital pulp or dentine. Traumamay be to non-permanent or permanent teeth, such as one or more molars,canine teeth, or incisors. Preferably, treatment of the subject withamelogenin and PGA composition disclosed herein is performed on a toothcontaining vital pulp or asymptomatic pulp where the tooth has not beensubjected to a pulpectomy (complete removal of pulp) or pulpotomy(partial removal of pulp).

Teeth include any one of the 32 human teeth identified by the universalnumbering system as well as human baby teeth. A baby tooth may be cappedin order to provide time for a healthy, properly aligned permanent toothto erupt. Human subjects include both males and females and children andadults, for example; children less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, or 18 years old and adults of at least 18,19, 20, 21, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 or >100 years old.

Other subjects include non-human mammals or other animals having teeth,such as a dog, cat, horse, or other domesticated or wild animals, suchas gorillas, orangutans, chimpanzees and other simians, lions, tigers,and cheetahs and other cats, bears, polar bears, pandas, elephants,zebras, giraffes, meerkats, goats, sheep, llamas, camels, etc. The term“tooth” as used herein also includes tusks, such as those of elephants,wild pigs and walruses. Preferably, the amelogenin used in thecomposition disclosed herein is a homologous amelogenin derived from thesame species of animal as the subject being treated, though in someembodiments, a xenogenic amelogenin such as one having at least 90, 95or 99% sequence identity to amelogenin of the subject being treated, maybe used due to the highly conserved nature of amelogenins.

Disinfection/Cleaning. Exposed dental surfaces may be cleaned and/ordisinfected, for example, by removal of decayed enamel or dentine, witha sterile or an antiseptic wash, sonic energy, UV or light exposure, ormechanically by complete or partial extraction prior to treatment withamelogenin and PGA. Damaged, unstable, or carried enamel or dentin canremoved prior to application of the composition of the inventionpreferably without aggravating any breach in the pulp chamber.

Direct and indirect capping. The method as disclosed herein may be usedfor both direct and indirect pulp capping. Pulp capping is a dentalprocedure used to prevent the death of dental pulp after an exposure ornear exposure, by placing a protective material or dressing over thepulp. This exposure may be due to a mechanical trauma after an accidentor tooth fracture such as a fractured or broken tooth, infected tooth ordeep cavity; or the pulp may be exposed during a cavity cuttingprocedure in the dental office or as a result of repeated dentalprocedures.

Indirect capping with a composition as disclosed herein is typicallyused for treating a tooth without any exposed pulp tissue, for example,when bacterial decay has reached near to the pulpal chamber, but notpenetrated it, and causes inflammation or threatens the survival of thetooth. Direct capping procedure is employed where pulp has already beenexposed or almost exposed and the composition as disclosed herein isapplied over the exposed or nearly exposed pulp tissue such as on thecrown of a tooth. A direct pulp cap is usually done on permanent teethwhen the removal of deep decay results in exposing the pulp. Insituations where the pulp is vital and healthy, capping exposed pulptissue with an amelogenin and PGA composition as disclosed herein, andproviding a good seal with the filling material may solve the problemand prevent the need for further endodontic treatment.

In some embodiments, the amelogenin and PGA composition of the inventionmay be loaded into separate syringes or other devices for administeringdental compositions and a delivered in to a tooth through a singlenozzle or needle that mixes them. In other embodiments, the amelogeninand vehicle are premixed and the administered as a mixed compositioninto or over a tooth. In some embodiments, the method of the inventiondoes not involve dispensing the amelogenin composition to near cutcervical enamel, to the root apex, or to periapical tissues around theroot apex.

In some embodiments, after capping a tooth with the composition asdescribed herein, the material is permitted to set for a period of 5,10, 15, 30, 45, 60, 90, 120 or 180 minutes prior to completion of thedental procedure, for example, by placement of a permanent restorative,and dismissal of the patient.

Amelogenin. As used herein, this term unless otherwise specifiedincludes human and non-human unprocessed, processed full-lengthamelogenin without its signal peptide, as well as amelogenin fragmentssuch as those resulting from the action of enzymes or proteases.Amelogenin M180 is a form of murine amelogenin. Representativeamelogenin nucleic acid/amino acid sequences include those of humanAMELX (SEQ ID NOS: 1 and 2), human AMELY (SEQ ID NOS: 3 and 4) andmurine amelogenin (SEQ ID NOS: 5 and 6).

Natural amelogenins are synthesized from alternatively spliced mRNAs,ranging in size from 5 to 28 kDa. These proteins are further processedat both their C- and N-termini by matrix metalloproteinase 20 (MMP20),to form many smaller peptide fragments, including a 23-kDa amelogenin(Amg23), a 5-kDa tyrosine-rich amelogenin peptide (TRAP), and a 5.4-kDaleucine-rich amelogenin peptide (LRAP). The Amg23 and TRAP are formedfrom a full-length amelogenin (Amg25), while the LRAP is generated froman alternatively spliced LRAP precursor, description of amelogeninfragments and proteases acting on amelogenin are incorporated byreference to Fincham et al., Biosci Rep. 1981 October; 1(10):771-8;Gibson, et al., Biochem Biophys Res Commun. 1991 Feb. 14;174(3):1306-12; Fincham and Moradian-Oldak, Biochem Biophys Res Commun.1993 Nov. 30; 197(1):248-55; and Connect Tissue Res. 1995;32(1-4):119-24; or Moradian-Oldak et al., Arch Oral Biol. 1994 August;39(8):647-56.

The sequence of naturally produced amelogenin is typically divided intothree prominent amino acid domains: a hydrophobic tyrosine-richN-terminal domain, called the tyrosine-rich amelogenin peptide (TRAP);the central proline-rich region, which is hydrophobic and primarilycomposed of X-Y-proline (where X and Y are often glutamine) repeatmotifs; and the hydrophilic C-terminal domain. The N- and C-terminalregion amino acid sequences are highly conserved among mammalianspecies, suggesting that these segments play important roles in enameldevelopment and mineralization. The native amelogenin contains a singlephosphate group on serine-16 that is presumed to be involved inamelogenin-calcium phosphate interactions and contribute to the abilityof amelogenin to stabilize the precursor amorphous calcium phosphate.The primary sequence is enriched with disorder-promoting residues, suchas Pro (P), Gln (Q), Glu (E), Arg (R), and Lys (K), leading to theintrinsically labile nature of the protein.^(4,5). Amelogenin belongs tothe class of intrinsically disordered or unstructured proteins (IDPs).Unlike folded proteins, IDPs lack regular secondary or tertiarystructure but are capable of transforming into a folded state followinginteractions with their targets and as part of their overall function.Full-length recombinant amelogenin molecules can spontaneouslyself-assemble into nanospheres under pH=8 conditions in the absence ofcalcium-phosphate. The inventors believe that the nanospheres wereformed through intermolecular hydrophobic interactions when thehydrophilic segment of each molecule was exposed on the surface of thenanospheres.—The nanospheres were then proposed to be the basicstructural entities of the developing enamel extracellular matrix and toplay a crucial function in enamel biomineralization.

Recombinant amelogenins for incorporation into an amelogenin and PGAcomposition can be synthesized from cDNA corresponding to the RNAsencoding amelogenin and its fragments by methods well known in the art,such as the cDNA cloning procedures described by Green, et al.,Molecular Cloning: A Laboratory Manual (Fourth Edition, Cold SpringHarbor Press. 2014 (incorporated by reference) or using commerciallyavailable cloning kits, such as PCR cloning kits and reagents and othermolecular biological materials, available from ThermoFisher Scientific(https://_www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-molecular-cloning.html?skwcid=AL!3652!10!76759715554332!76759737462825&ef_id=XEeKuQAAEqiq9BGW:20190503151725:s, last accessed May 3, 2019, incorporated byreference). In one embodiment recombinant poly-(histidine)-taggedproteins were expressed and recovered. These, recombinant amelogeninproteins were prepared with the expression vector pQE30 (QIAGEN Inc.,Valencia, Calif.) and purified with a poly-(histidine) amino terminus,as previously described by Moradian-Oldakm et al., J, Struct. Biol.131(1):2737 (2000) and Viswanathan, et al., J. Periodontaol74(10):1423-31 (2003) both incorporated by reference. Recombinantpoly-histidine-tagged murine 180 amino acid long amelogenin (rp(H) M180)is identical to the authentic mouse full-length amelogenin except forthe inclusion of an amino-terminal peptide sequence (RGSHHHHHHGS)(SEQ IDNO: 7) used for affinity purification of the bacterially producedprotein. The entire DNA fragment within the pQE30 vector was subjectedto nucleotide sequence determination to ensure that no errors weregenerated during the DNA amplification procedures and that the constructwas correctly engineered. Recombinant proteins were prepared andpurified using nickel-nitrilotriacetic acid (Ni-NTA) metal-affinitychromatography matrices (Qiagen, Valencia, Calif.).

The major fraction of the enamel matrix proteins is composed ofamelogenins, a family of hydrophobic proteins that account for more than90% of the organic constituents of the enamel matrix. Unless otherwisespecified the term amelogenin includes full-length amelogenin as well asprocessed forms of amelogenin such as amelogenin fragments as well aslonger proteins such as an amelogenin comprising a signal peptide, aFLAG or HIS or other tag, or another exogenous amino acid sequence. Italso includes variant amelogenin proteins such as those having at least90, 95, 99 or <100% sequence identity to an amelogenin amino acidsequence disclosed herein as well as variants having 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more deletions, additions or substitutions of amino acidresidues to a known amelogenin sequence.

BLASTN may be used to identify a polynucleotide sequence having at least70, 75, 80, 85, 90, 95, 96, 97, 98, 99, <100, or and 100% (or anyintermediate %) sequence identity to a reference polynucleotide, such asa nucleic acid, such as cDNA or mRNA encoding an amelogenin as disclosedherein. A representative BLASTN setting optimized to find highly similarsequences uses an Expect Threshold of 10 and a Wordsize of 28, maxmatches in query range of 0, match/mismatch scores of 1/-2, and lineargap cost. Low complexity regions may be filtered/masked. Defaultsettings are described by and incorporated by reference tohttp://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome (last accessedMay 3, 2019).

BLASTP can be used to identify an amino acid sequence having at least70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96, 97.5%, 98%, 99%, <100%or 100% (or any intermediate %) sequence identity or similarity to areference amino acid using a similarity matrix such as BLOSUM45,BLOSUM62 or BLOSUM80 where BLOSUM45 can be used for closely relatedsequences, BLOSUM62 for midrange sequences, and BLOSUM80 for moredistantly related sequences. Unless otherwise indicated a similarityscore will be based on use of BLOSUM62. When BLASTP is used, the percentsimilarity is based on the BLASTP positives score and the percentsequence identity is based on the BLASTP identities score. BLASTP“Identities” shows the number and fraction of total residues in the highscoring sequence pairs which are identical; and BLASTP “Positives” showsthe number and fraction of residues for which the alignment scores havepositive values and which are similar to each other. Amino acidsequences having these degrees of identity or similarity or anyintermediate degree of identity or similarity to the amino acidsequences disclosed herein are contemplated and encompassed by thisdisclosure. A representative BLASTP setting that uses an ExpectThreshold of 10, a Word Size of 3, BLOSUM 62 as a matrix, and GapPenalty of 11 (Existence) and 1 (Extension) and a conditionalcompositional score matrix adjustment. Default settings for BLASTP aredescribed by and incorporated by reference to:http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome(last accessed May 3, 2019).

Amelogenin compositions. One or more of amelogenin or its proteinfragments may be incorporated into an amelogenin and PGA compositionaccording to the invention.

Typically, the composition of the invention will comprise full-lengthamelogenin, though in some embodiments it will comprise or consistessentially of an amelogenin fragment or a mixture of full-length andamelogenin fragments in the same weight proportion to PGA as describedfor full length amelogenin. Such a mixture may contain 0.1, 0.2, 0.5, 1,2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, 99, 99.9 to <100% offull-length amelogenin and 99.9, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5,2, 1, 0.5, 0.2, 0.1 to <0.1 wt % one or more amelogenin fragments. Inother embodiments, the composition may comprise full-length recombinantamelogenin and MMP20 or another protease that processes full-lengthamelogenin into smaller fragments.

The amelogenins, which are the hydrophobic constituents of the enamelmatrix proteins, aggregate and become almost insoluble at physiologic pHand temperature. They can be dissolved in an acidic or alkaline pHenvironment and at low temperature. A suitable formulation should thushave a non-neutral pH and allow for gradual reprecipitation of thematrix when physiologic conditions are re-established.

An amelogenin composition according to the invention may contain one ormore pharmaceutically acceptable excipients and be in form of a solid,semi-solid or liquid composition including powders, granules,granulates, capsules, agarose or chitosan beads, tablets, pellets,microcapsules, microspheres, nanoparticles, or freeze-dried powders,granules, granulates or pellets, gels, hydrogels, pastes, solutions,dispersions, suspensions, emulsions, or mixtures.

The compositions may be formulated according to conventionalpharmaceutical practice, see, e.g., “Remington's PharmaceuticalSciences” and “Encyclopedia of Pharmaceutical Technology”, edited bySwarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988. Apartfrom the amelogenin, a pharmaceutical composition for use according tothe invention may comprise pharmaceutically acceptable excipients. Apharmaceutically acceptable excipient is a substance which issubstantially harmless to the individual to which the composition is tobe administered. Such an excipient normally fulfils the requirementsgiven by the national health authorities. Official pharmacopoeias suchas e.g. the British Pharmacopoeia, the United States of AmericaPharmacopoeia and The European Pharmacopoeia set standards forpharmaceutically acceptable excipients. The choice of a pharmaceuticallyacceptable excipient in a composition for use according to the inventionand the optimum concentration thereof cannot generally be predicted andmust be determined on the basis of an experimental evaluation of thefinal composition. However, a person skilled in the art ofpharmaceutical formulation can find guidance in “Remington'sPharmaceutical Sciences”, 21^(st) Edition, Mack Publishing Company,Easton, 2005. The pharmaceutically acceptable excipients may includesolvents, buffering agents, preservatives, chelating agents,antioxidants, stabilizers, suspending agents and gel-forming agents.Examples of solvents are water, alcohols, or other hydrophilic oretheric solvents such as weak acids with a pH of about 5.5-6.0facilitating the subsequent application of filling materials in thetooth. Examples of buffering agents are citric acid, acetic acid,tartaric acid, lactic acid, hydrogenphosphoric acid, and diethylamine.Suitable examples of preservatives for use in compositions are parabens,such as methyl, ethyl, propyl p-hydroxybenzoate, butylparaben,isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid,benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDMhydantoin, iodopropynyl butylcarbamate, benzalconium chloride, andbenzylalcohol, or mixtures of preservatives. Examples of antioxidantsare butylated hydroxy anisole (BHA), ascorbic acid and derivativesthereof, tocopherol and derivatives thereof, cysteine, and mixturesthereof. Examples of suspending agents are celluloses and cellulosederivatives such as carboxymethyl cellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystallinecellulose (e.g. Avicel® RC 591), carrageenan, acacia gum, arabic gum,tragacanth, and mixtures thereof. Examples of gel bases orviscosity-increasing agents are liquid paraffin, polyethylene, fattyoils, colloidal silica or aluminum, zinc soaps, glycerol, propyleneglycol, tragacanth, carboxyvinyl polymers, magnesium-aluminum silicates,Carbopol™, hydrophilic polymers such as starch or cellulose derivativessuch as carboxymethylcellulose, hydroxyethylcellulose and othercellulose derivatives, water-swellable hydrocolloids, carragenans,hyaluronates, and alginates including propylene glycol alginate.Examples of powder components include alginate, collagen or lactose.Normally, powders intended for application on dental pulps must besterile and the particles present must be micronized. Examples of otherexcipients are polymers such as carmelose, sodium carmelose,hydroxypropylmethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, pectin, xanthan gum, locust bean gum, acaciagum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearates,cetanyl glucoside, collagen, carrageenan, hyaluronates and alginates andchitosans. Suitable compositions for use according to the invention mayalso be presented in the form of suspensions, emulsions or dispersions.Such compositions can contain the active enamel substance in admixturewith a dispersing or wetting agent, suspending agent, and/or one or morepreservatives and other pharmaceutically acceptable excipients. Suitabledispersing or wetting agents are, for example, naturally occurringphosphatides, e.g., lecithin, or soybean lecithin; condensation productsof ethylene oxide with a fatty acid, a long chain aliphatic alcohol, ora partial ester derived from fatty acids and a hexitol or a hexitolanhydride, for example polyoxyethylene stearate, polyoxyethylenesorbitol monooleate, or polyoxyethylene sorbitan monooleate.

Enamel matrix and other proteins. Other proteins which may beincorporated into an amelogenin and PGA composition as disclosed hereininclude enamelins, amelins, ameloblastin, sheathlin, tuftelins, tuftproteins, dentinsialoprotein, dentinsialophosphoprotein, serum proteins,and salivary proteins. The second largest component of the enamel matrixprotein is the enamelins. Enamelins have been found to contain serumproteins, and the more general term “non-amelogenin” is now commonlyused to describe this high molecular weight fraction, which includesproline-rich enamelin, tuftelin, and tuft proteins.

The composition of the invention may include, in addition to one or moreamelogenins, any of the non-amelogenin enamel matrix proteins, forexample, from 0.1, 0.2, 0.5, 1, 2, 5, 10 or >10 wt % based on the weightof the amelogenin composition.

Other ingredients. In some embodiments, the amelogenin and PGAcomposition of the invention will further include, or be further admixedwith, at least 0.01, 0.02, 0.05, or 0.1 wt % based on a total weight ofthe composition, a protease, such as MMP20, that processes amelogenin,or with at least 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 or 20 wt %agents such as one or more of tricalcium silicate, dicalcium silicate,tricalcium aluminate, tetracalcium aluminoferrite, calcium sulfate, andbismuth oxide, which are also ingredients in MTA. However, in manyembodiments, the composition as disclosed herein consists of, orconsists essentially of an amelogenin and PGA, without other cappingagents.

Alginates and thickeners. In a preferred embodiment, the amelogenincomposition described herein contains an alkylene glycol alginate, suchas propylene glycol alginate (“PGA”). In some embodiments, the alkyleneglycol alginate is formed from incomplete esterification of alginicacid, whereby some of the carboxyl groups of alginic acid or saltsthereof are esterified with an alkylene oxide, while the remainderremain free or are neutralized with an appropriate alkali base. Thealkylene glycol alginate may be a ‘low esterification type’, whereinless than 60%, preferably less than 50%, preferably less than 40% of thetotal number of carboxyl groups of alginic acid are esterified with analkylene oxide(s) such as propylene glycol, the remaining groups beingeither free or neutralized with a base. In preferred embodiments, thealkylene glycol alginate is a ‘high esterification type’ where at least60%, preferably at least 70%, preferably at least 75%, preferably atleast 80%, and up to 90%, preferably up to 85% of a total number ofcarboxyl groups present in alginic acid or salts thereof are esterifiedwith the alkylene oxide(s) to form the alkylene glycol alginate.

In some embodiments, the alkylene glycol alginate is formed using one ormore alkylene oxides having 2 to 6 carbon atoms, preferably 3 to 4carbon atoms, more preferably 3 carbon atoms. Suitable alkylene oxidesspecifically contemplated are ethylene oxide, propylene oxide,1,3-epoxypropane, 1,2-epoxybutane, 2,3-epoxybutane,1,2-epoxy-2-methylpropane, 1,2-epoxypentane, and 1,2-epoxyhexane,mixtures thereof, as well as any other alkylene oxide known by those ofordinary skill in the art as being appropriate for the preparation ofalkylene glycol alginates. Among these alkylene oxides, propylene oxideand butylene oxide are preferred, more preferably propylene oxide.

Propylene glycol alginate is an emulsifier, stabilizer, and thickenerused in food products. Chemically, propylene glycol alginate is an esterof alginic acid, which is derived from kelp. Some of the carboxyl groupsare esterified with propylene glycol, some are neutralized with anappropriate alkali, and some remain free. However, in alternativeembodiments, amelogenin may be combined at the same weight ratios asdisclosed herein for PGA with one or more other thickeners.

In preferred embodiments, the alkylene glycol alginate employed hereinis propylene glycol alginate (PGA). Propylene glycol alginate is a goodthickener which exhibits slight pseudoplastic behavior. Propylene glycolalginates are broken down into grades based on viscosity andesterification levels (i.e., the percentage of esterified carboxylgroups described heretofore), and any grade of propylene glycol alginatemay be utilized. Within the category of standard esterification types(e.g., less than 60% of carboxyl groups esterified), the propyleneglycol alginate may have the following grades (based on viscosity rangesof a 1 wt. % solution at 20° C.): “LVC” (70 to 170 mPa·s); or “HVC” (200to 600 mPa·s). Within the category of high esterification types (e.g.,60 to 90% of carboxyl groups esterified), the propylene glycol alginatemay have the following grades (based on viscosity ranges of a 1 wt. %solution at 20° C.): “LLV” (15 to 35 mPa·s); “NLS-K” (30 to 60 mPa·s);“LV” (60 to 100 mPa·s); “MV” (100 to 150 mPa·s); and “HV” (150 to 250mPa·s).

In preferred embodiments, the propylene glycol alginate utilized hereinis an “HV” grade, i.e., is of a high esterification type having aviscosity of at least 150 mPa·s, preferably at least 175 mPa·s,preferably at least 200 mPa·s, and up to 250 mPa·s, preferably up to 225mPa·s, preferably up to 215 mPa·s.

Propylene glycol alginates are available for example from Kimica Corp.under the tradename KIMILOID, for example, KIMILOID HV.

Most preferably, PGA is used in combination with amelogenin in acomposition according to the invention. However, in some alternativeembodiments, other thickeners may be used, especially thickeners whichare biodegradable or metabolizable. Other thickeners include alginicacid, sodium alginate, potassium alginate, ammonium alginate, calciumalginate, polysaccharides derived from red or brown algae; PEG, agar,carrageenan, locust bean gum, xanthan, acacia, pectin, gelatin, andvarious cellulosics, sulfonates, and saccharides.

Amelogenesis imperfecta. A subject may express an isotype of a normal orwild-type amelogeniin, which is associated with harder and/or notdiscolored teeth, or express a mutated form of amelogenin, such as onehaving T21I or P40T substitutions in the amelogenin sequence, which isassociated with amelogenesis imperfecta causing the tooth to behypomineralized and to have a brown discoloration. Such a subject mayhave a tooth partially restored, capped or otherwise treated using acomposition containing a normal wild-type form of amelogenin, a mixtureof wild-type and mutated amelogenin, or with a composition containing amutated form of amelogenin. For a subject expressing a non-wild-typeform of amelogenin, such as a mutant form that forms softer ordiscolored teeth or a form of amelogenin associated with amelogenesisimperfecta, direct or indirect capping may be performed using acomposition comprising wild-type amelogenin in order to compensate fornegative effects of the mutant form of amelogenin. For example, asubject suffering from amelogenesis imperfecta can have a soft ordiscolored tooth hardened and whitened by capping with a wild-typeamelogenin. A mixture of wild-type and mutant amelogenin may contain aweight ratio of wild-type amelogenin to mutant amelogenin of 100:1,50:1, 20:1, 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, 1:10, 1:20, 1:50 or 1:100. Insome embodiments, the amelogenin is full-length amelogenin. In otherembodiments, at least one form of processed or truncated full-lengthamelogenin may be used.

Restoration materials. After application of the amelogenin-PGAcomposition of the invention on portions of teeth containing open dentintubules or that expose pulp, a restorative is added to seal the tooth.It cures extremely hard and is very wear resistant. As a true glassionomer, it chemically bonds to tooth structure, has a tooth-likecoefficient of thermal expansion and releases significant levels ofrechargeable fluoride. Other restorative materials include preciousmetal alloys such as gold, gold alloys, gold-platina alloy,silver-palladium alloy, cobalt-chrome alloy, nickel-chroma alloy;amalgam, such as silver amalgam or high copper dental amalgam; directgold; composite resin, such as BISMA or UDMA or TEGMA; glass ionomercement; porcelain, such as glass-bonded porcelain, lithium disilicateglass ceramic and phase stabilized zirconia. One example, of arestorative is GC Fuji IX GP which is a fluoride releasing alternativeto expensive compomers and composites and in many cases, amalgam. Insome embodiments, calcium hydroxide and bioceramic material are used.

Examples

Exposure of vital dental pulp, either accidentally or by design, is acomplication commonly seen in dental practice. Preserving the dentalpulp or part of it in a healthy state is important in treating teethwith exposed vital pulp. To test the effects of exposing vital dentalpulp to the amelogenin and PGA composition of the invention, thefollowing experiments were conducted.

Recombinant amelogenin was produced essentially as described byMoradian-Oldak J, Paine M L, Lei Y P, et al. Self-assembly properties ofrecombinant engineered amelogenin proteins analyzed by dynamic lightscattering and atomic force microscopy. J Struct Biol 2000; 131:27-37;Viswanathan H L, Berry J E, Foster B L, et al. Amelogenin: a potentialregulator of cementum-associated genes. J Periodontol 2003; 74:1423-31(each incorporated by reference). Recombinant poly-histidine-taggedmurine 180 amino acid long amelogenin (rp(H) M180) is identical to theauthentic mouse full-length amelogenin except for the inclusion of anamino-terminal peptide sequence (RGSHHHHHIHGS)(SEQ ID NO: 7) used foraffinity purification of the bacterially produced protein. Briefly,recombinant amelogenin proteins comprising a polyhistidiine aminoterminus were prepared with the expression vector pQE30 (QIAGEN Inc,Valencia, Calif.) and affinity purified using nickel nitrilotriaceticacid metal-affinity chromatography matrices (QIAGEN Inc).

Propylene glycol alginate vehicle as used in these experiments contained30 mg of propylene glycol aliginate in 1 cm³ of saline. Approximately 1mL propylene glycol alginate vehicle was mixed with 30 mg recombinantamelogenin protein powder (rp[H] M180) 15 minutes before use. Themixture was placed on the exposed pulp to be compared with MTA cappingmaterial. PGA powder concentration was determined by the manufacturer ofr-amelogenin.

After 10 days various differences between teeth treated with thecomposition of the invention, containing recombinant amelogenin and PGA,and the conventional MTA treatment were assessed. Results are shown inTable 1 below, FIG. 10 which graphically depicts the significantimprovements in tooth vitality provided by the invention, and by FIGS. 1and 2 which show the improved histological appearance of teeth treatedwith amelogenin and PGA compared to those treated with MTA shown byFIGS. 5 and 6.

The histological preparation of the samples was done in the followingsteps: Samples were kept in 10% neutral formalin to achieve completefixation, then washed for 24 hours in order to remove the remnants ofthe fixative solution, and decalcified by 5% trichloroacetic acid. Afterdecalcification all samples were again washed for 24 hours to removeexcess acid. All samples were dehydrated using different grades ofalcohol 50%, 70%, and 90% and absolute, however dehydration timedepended on the density and size of each sample, lastly absolute alcoholwas changed two Limes.

Samples were transferred from absolute alcohol to xylene until thecomplete replacement of the tissue by the xylene when the tissue iscompletely replaced with xylene paraffin ready to be infiltrated. Thensamples were removed from the xylene and placed in melted dishes ofparaffin wax and placed into an oven at a 60 C for 2-3 hours. Aftercomplete infiltration of the samples by paraffin, they were placed inthe centre of paraffin wax box. Each tooth was sectioned in the middleof the prepared cavity bucco-lingually

Serial bucca-lingual sections of the cavity were obtained using amicrotome. All sections were mounted on clean slides and placed in theoven at certain temperature and then deparafanized by passing them intoa two changes of xylene then two changes of absolute alcohol, thenplaced in 50%, 70, and 90? grades of alcohol to ensure completedehydration, then passed through distilled water for proper staining ofthe samples. Serial sections were stained by hernatoxylin and eosinstain and trichrome stain.

Serial sections were analysed under light microscope associated withdigital camera. Each specimen was observed for: (I) calcified tissuebarrier formation, complete or incomplete; (2) presence or absence ofpulp tissue; (3) condition of the present pulp, normal or altered; (4)presence of pulp inflammation; and (5) presence of pulp calcification.Analysis of the inflammatory reaction was done according to differentgiven scores:

Score 0: Absence of inflammatory cells.

Score 1: Mild scattering of inflammatory cells with no structuraldamage.

Score 2: Moderate focal accumulations of inflammatory cells, no tissuenecrosis with some disruption of the structure.

Score 3: Severe extensive inflammatory cell infiltrate with Replacementof tissues and abscess formation.

Analysis of dentin bridge formation was done according to given grades:

Grade1: complete dentin bridge formation

Grade2: the hard tissues are moderately formed.

Grade 3: hard tissues are slightly formed.

Grade4: lack of hard tissue deposition.

All samples were statistically analysed.

TABLE 1 Hard tissue formation, pulp condition and inflammatory reactionafter using Amelogenin and MTA as pulp capping material after 10 daysobservation period. Variables MTA Amelogenin Total number of teeth 20(50%) 20 (50%) 1-Dentin bridge formation a-complete (grade-1) 0.0 (0.0%)0.0 (0.0%) b-hard tissues moderately formed (grade 2) 0.0 (0.0%) 16(80%) c-hard tissues slightly formed (grade 3) 11 (55%) 4 (20%) d-lackof hard tissues formed (grade 4) 9 (45%) 0.0 2-Pulp status a-presence ofodontoblasts 20 (100%) 20 (100%) b-presence of calcifications 0.0 (0.0%)4 (20%) c-presence of dilated blood vessels 20 (100%) 20 (100%)d-presence of dilated lymphatic vessels 0.0 (0.0%) 0.0 (0.0%) e-presenceof pulp tissue filling the whole pulp chamber 5 (25%) 20 (100%)f-presence of pulp tissue incompletely filling the whole pulp chamber 15(75%) 0.0 (0.0%) and/or presence of voids g-empty pulp space 0.0 (0.0%)0.0 (0.0%) 3-Presence of inflammatory cells in pulp a-absence ofinflammatory cells (score 0) 0.0 (0.0%) 0.0 (0.0%) b-mild scattering ofinflammatory cells with no structural damage 20 (100%) 20 (100%)(score 1) c-moderate, focal accumulation of inflammatory cells, tissue0.0 (0.0%) 0.0 (0.0%) necrosis with some disruption of the structure(score 2) d-severe extensive inflammatory cell infiltrate withreplacement of 0.0 (0.0%) 0.0 (0.0%) tissue and abscess formation (score3)

As shown by Table 1 above, after 10 days, compared to the conventionalMTA treatment, the recombinant amelogenin-PGA composition of theinvention formed significantly more grade 2 and grade 3 hard dentinbridge tissues and calcifications than the MTA composition. It alsoproduced significant improvements in the vital pulp tissue which wasvoid free in 100% of the teeth treated with the amelogenin-PGAcomposition compared to teeth treated with MTA in which only 25% of thepulp chambers were void free. As shown by these results the method asdisclosed herein, using a mixture of recombinant amelogenin and PGA,induced the formation of a tertiary dentine mineralized barrier thatefficiently sealed-in exposed dental pulp and also preserved thevitality of the tooth by preserving vital pulp in the pulp chamber. Thismethod effectively extends the life expectancy of damaged teeth.

After 2 months various differences between teeth treated with thecomposition of the invention, containing recombinant amelogenin and PGA,and the conventional MTA treatment were again assessed. Results areshown in Table 2 below and by FIGS. 3 and 4 which show the improvedhistological appearance of teeth treated with amelogenin and PGAcompared to those treated with MTA shown by FIGS. 7, 8 and 9. As shownby these figures, while teeth capped with MTA instead of amelogenin-PGAcan show formation of a dentin bridge, the underlying pulp tissueexhibits undesirable changes. These include dilated blood vessels andlack of contact between the MTA capping material and the underlying pulpresulting in formation of voids in the pulp tissue. In some instances,the pulp shows complete degeneration. In contrast, after 2 months, thereare no voids in the teeth capped with amelogenin-PGA.

The histology of teeth capped with amelogenin-PGA show the formation ofdentin bridge as early as 10 days placement of the material (FIGS. 1 and2) and a thick dentin bridge by 2 months (see FIGS. 5 and 6). Theunderlying pulp is always in contact with amelogenin cap and theauthentic pulp tissue structure is preserved in all time periods.

TABLE 2 Hard tissue formation, pulp condition and inflammatory reactionafter using Amelogenin and MTA as pulp capping material after two monthsobservation period. Variables MTA Amelogenin Total number of teeth 20(50%) 20 (50%) 1-Dentin bridge formation a-complete (grade 1) 11 (55%)20 (100%) b-hard tissues moderately formed (grade 2) 8 (40%) 0.0 (0.0%)c-hard tissues slightly formed (grade 3) 0.0 (0.0%) 0.0 (0.0%) d-lack ofhard tissues formed (grade 4) 1 (5%) 0.0 (0.0%) 2-Pulp status a-presenceof odontoblasts 19 (95) 20 100%) b-presence of calcifications 4 (20%) 3(15%) c-presence of dilated blood vessels 20 (100%) 8 (40%) d-presenceof dilated lymphatic vessels 0.0 (0.0%) 0.0 (0.0%) e-presence of pulptissue filling the whole 0.0 (0.0%) 20 (100%) pulp chamber f-presence ofpulp tissue incompletely filling 16 (80%) 0.0 (0.0%) the whole pulpchamber and/or presence of voids g-nearly empty pulp space 4 (20%) 0.0(0.0%) h-authentic pulp architecture not preserved 0.0 (0.0%) 0.0 (0.0%)3-Presence of inflammatory cells in pulp a-absence of inflammatory cells(score 0) 19 (95%) 20 (100%) b-mild scattering of inflammatory cellswith 1 (5%) 0.0 (0.0%) no structural damage (score 1) c-moderate, focalaccumulation of 0.0 (0.0%) 0.0 (0.0%) inflammatory cells, tissuenecrosis with some disruption of the structure (score 2) d-severeextensive inflammatory cell infiltrate 0.0 (0.0%) 0.0 (0.0%) withreplacement of tissue and abscess formation (score 3)

As shown by Table 2 above, after 2 months compared to the conventionalMTA treatment, the recombinant amelogenin-PGA composition of theinvention form significantly 100% grade 1 hard dentin bridge formationcompared to only 55% in teeth treated with the MTA composition. 100% ofthe teeth treated with the amelogenin-PGA composition showed pulp tissuefilling the whole pulp chamber compared to 0% for teeth treated withMTA. Only 40% of the teeth treated with the amelogenin-PGA compositionstill exhibited dilated blood vessels compared to 100% for the MTA.

Terminology. Terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

The headings (such as “Background” and “Summary”) and sub-headings usedherein are intended only for general organization of topics within thepresent invention, and are not intended to limit the disclosure of thepresent invention or any aspect thereof. In particular, subject matterdisclosed in the “Background” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

Links are disabled by deletion of http: or by insertion of a space orunderlined space before www. In some instances, the text available viathe link on the “last accessed” date may be incorporated by reference.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper”, “in front of” or “behind” and the like, may be used herein forease of description to describe one element or feature's relationship toanother element(s) or feature(s) as illustrated in the figures. It willbe understood that the spatially relative terms are intended toencompass different orientations of the device in use or operation inaddition to the orientation depicted in the figures. For example, if adevice in the figures is inverted, elements described as “under” or“beneath” other elements or features would then be oriented “over” theother elements or features. Thus, the exemplary term “under” canencompass both an orientation of over and under. The device may beotherwise oriented (rotated 90 degrees or at other orientations) and thespatially relative descriptors used herein interpreted accordingly.Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal”and the like are used herein for the purpose of explanation only unlessspecifically indicated otherwise.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference,especially referenced is disclosure appearing in the same sentence,paragraph, page or section of the specification in which theincorporation by reference appears.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

1. A method for treating exposed dental pulp of a tooth in a subjecthaving a damaged tooth, comprising: applying a treatment compositioncomprising amelogenin and propylene glycol alginate (“PGA”) to a surfaceof exposed pulp of the tooth; and applying a permanent dentalrestorative on top of the treatment composition present on the exposedenamel, wherein the treatment composition comprises 30 mg amelogeninprotein powder in mixture with 1 ml of a combination of propylene glycolalginate and a saline matrix. 2-3. (canceled)
 4. The method of claim 1,further comprising, after applying the composition, applying a permanentdental restorative on top of the applied composition on the same day.5-6. (canceled)
 7. The method of claim 1, wherein the amelogenincomprises an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 2, 4 or 6 or a sequence that has atleast 95% sequence identity to SEQ ID NOS: 2, 4 or 6 or a fragmentthereof having at least 30 contiguous amino acid residues.
 8. The methodof claim 1, wherein the subject is a human and the amelogenin compriseshuman amelogenin or a fragment thereof having at least 30 contiguousamino acid residues of the human amelogenin.
 9. (canceled)
 10. Themethod of claim 1, wherein the treatment composition consistsessentially of recombinant human amelogenin, the saline and thepropylene glycol alginate.
 11. The method of claim 1, wherein thetreatment composition further comprises a remineralization agentselected from the group consisting of a material providing calcium ions,a material providing phosphorous ions, and a material providing fluorideions.
 12. The method of claim 1, wherein said treatment compositionfurther comprises at least one non-amelogenin enamel matrix protein. 13.The method of claim 1, wherein said treatment composition furthercomprises at least one bacteriostatic or bactericidal agent. 14-15.(canceled)
 16. The method of claim 1, wherein the subject is sufferingfrom amelogenesis imperfecta and said composition comprises a wild-typeamelogenin. 17.-20. (canceled)